Involvement of an Aspergillus fumigatus putative sphingolipid-synthesis related protein OrmA in antifungal azole stress responses
Previous studies have identified that the sphingolipid, which is a major structural component of eukaryotic cytoplasmic membrane, is involved in many important biological processes, including cell metabolism and stress tolerance response, etc. It has been reported that there are two sphingolipid-synthesis related protein Orm1p and Orm2p in Saccharomyces cerevisiaes, which negatively regulate activities of the serine palmitoyl transferase that is the first speed-limit enzyme of sphingolipid synthesis.
In this study, through genome-wide homolog search analysis, we found that the A. fumigatus genome only contains one Orm homolog, referred as OrmA. Deletion of ormA causes hypersensitivity to azole while overexpression of OrmA shows the azole resistance. Moreover, Western blotting results indicate that OrmA protein expression could be induced by azole antifungals in a dose dependent way. These data suggest that OrmA in A. fumigatus is required for responding to the azole stress. In addition, rescued phenotypes for susceptibility to azole drugs by adding an sphingolipid synthesis inhibitor-myriocin in ormA-defective strains combined with data for deletion of ormA leads to significant increased main aacomponents of sphingolipid ceramide, suggesting that OrmA may work as a negative regulator for the sphingolipid synthesis.
Further mechanism analysis verified that OrmA is involved in drug susceptibility through affecting endoplasmic reticulum stress responses in an unfolded protein response pathway (UPR) HacA-dependent way. Our data suggest that endoplasmic reticulum stress caused by azole drugs could stimulate HacA activation accompanied with inducing the increased expression of OrmA so that affecting the sensitivity of azole drugs by regulating the synthesis of sphingolipid. Most importantly, virulence tests demonstrated that OrmA deletion caused a significantly decreased virulence in immunosuppressive mice model. Our findings suggest that the unexplored sphingolipid metabolism pathway in A. fumigatus plays important roles for fungal virulence and azole susceptibilities and it may be used as new antifungal drug targets.